抑癌基因PCDH10调控人胰腺癌细胞端粒酶活性作用及机制研究

Telomerase has been shed light on a broad spectrum of research fields including normal ageing, various forms of cancer, stem cells and age-related disease such as heart disease and diabetes because it emerged as a central player in cellular processes, such as cellular senescence, aging, DNA damage/repair, cellular immortalization. hTERT (Human telomerase reverse transcriptase) is not only the catalytic subunit of the telomerase, but also the rate-limiting determinant of telomerase activity. Considering that protein-protein interactions play important roles in almost all the events that take place in cells, in our previus study we found PCDH10 (Procadherin 10) interacted with hTERT through Co-IP-MS (Co-immunoprecipitation and mass spectrum) strategy fo the first time, then we identified PCDH10 bound specifically with hTERT in vivo by Co-IP and western blot analyses. Fourther we up-expressed the specific expression of PCDH10 by transfecting over-expression adenovirus expression vector into human pancreatic cancer cell (HS 776T) and demonstrated that this interaction by PCDH10-mediated immuno-precipitation inhibited telomerase activity but with no change of hTERT expression at both mRNA and protein levels. So we speculate that the interaction between PCDH10 and hTERT may inhibite the activity of telomerase directly. To address these issues, in our present study we will try to detect the common subcellular location of PCDH10 and hTERT and their interaction site by GST pull-down, and then to construct the PCDH10 binding site deletant of hTERT (hTERTΔ) and the hTERT binding site deletant of PCDH10 (PCDH10Δ), and to transfect them into HS 776T. Finally we will detect the expressions of PCDH10 and hTERT by using RT-PCR, western blotting, determine telomerase activitiy by TRAP and observe the morphology and growth of tumor cells finally. To sum up, we will validate the role and molecular machanism of a novel tumor suppressor gene PCDH10 on telomerase activity. These findings will likely not only contribute to further investigations of the machanism of telomerase on telomere length maintenance, cellular senescence and immortalization, oncogenesis and potentially new cellular functions, but also impact on our ability to manipulate telomerase for cancer-therapeutic, age-related diseases and tissue-engineering applications.

端粒酶与肿瘤发生和发展密切相关，降低端粒酶活性导致肿瘤细胞凋亡和衰老。hTERT是端粒酶活性的必需和限速成分，本课题组前期利用Co-IP&MS筛选hTERT相互作用蛋白抑癌基因PCDH10，并在体内证实两者的相互作用，这种作用对HS776T细胞端粒酶活性起抑制作用，但对hTERT表达没有影响。我们推测PCDH10通过与hTERT直接作用，抑制端粒酶活性，进而抑制肿瘤细胞生长、促进肿瘤细胞凋亡、降低肿瘤细胞侵袭转移。本课题拟在前期研究工作基础上，通过GSTpull-down体外明确PCDH10与hTERT的相互作用位点，并构建两者相互作用位点的缺失体，观察其对端粒酶活性和肿瘤细胞生物学特性的变化。从而明确 PCDH10与hTERT 相互作用对端粒酶活性的调控作用和机制，理论上为完善端粒酶活性调控机制提供新的实验依据和研究方向，实践上为端粒酶在肿瘤的发生、预防、诊疗方面提供新的思路和线索。

端粒酶与和肿瘤密切相关，但有关端粒酶活性构成和调控机制等基本问题尚未完全明了，阻碍端粒酶应用性研究。人端粒酶催化亚单位（hTERT）是端粒酶活性的必需和限速成分，其调控因子间接影响端粒酶活性。因此，以hTERT为作用靶点，深入研究端粒酶活性调节机制，有助于认识端粒酶在肿瘤发生发展中意义，进一步探索抗肿瘤有效途径。.课题组曾在两项国家自然科学基金资助下，通过酵母双杂交筛选获得一组hTERT相互作用蛋白，初步探索了该组蛋白对端粒酶活性和细胞生物学特性影响，为本课题研究打下有利基础。本课题前期研究中，我们首次在正常人睾丸组织中利用免疫共沉淀（Co-IP）联合串联芯片液相质谱，自行鉴定出新的hTERT相互作用蛋白：原钙粘附蛋白10 （PCDH10），并在体内利用Co-IP进一步证实PCDH10与hTERT具有相互作用。结合PCDH10是新发现的抑癌基因，而hTERT与肿瘤细胞永生化密切相关，提示其可能对端粒酶活性有影响，通过预实验表明体外增强PCDH10表达，其与hTERT相互作用可抑制胰腺癌细胞端粒酶活性，但对hTERT表达没有影响。此课题中我们首先在体外诱导表达PCDH10蛋白，采用Fortebio Octet’system 进一步验证PCDH10与hTERT具有相互作用；再次通过在胰腺癌细胞中过表达PCDH10，阐明PCDH10对hTERT表达无影响，而它与hTERT相互作用抑制端粒酶活性；最后明确PCDH10在体外具有抑制肿瘤细胞生长，降低肿瘤细胞迁移和侵袭等细胞生物学效应。.通过此课题研究，我们首次阐明PCDH10是hTERT相互作用蛋白，进一步取得直接证据，明确两者相互作用对端粒酶活性的调控作用，为完善端粒酶活性调控机制提供新的实验依据和研究方向；并探索PCDH10抑癌机制：抑癌基因PCDH10在多种肿瘤细胞和组织中低表达，造成与hTERT相互作用减弱，进而对端粒酶活性抑制作用减弱，使得肿瘤细胞生长和侵袭转移；反之，在肿瘤细胞中外源性过表达PCDH10，增强其与hTERT的直接相互作用，进而加强抑制端粒酶活性作用，从而抑制肿瘤细胞生长，促进肿瘤细胞凋亡，降低肿瘤细胞侵袭转移。目前国内外未见类似报道，这可能是PCDH10抑癌的重要机制之一，并且PCDH10很可能作为端粒酶抑制剂为抗肿瘤治疗提供新的靶点，为端粒酶在肿瘤发生、预防、诊断及治疗等方面提供新的思路和线索。